The present invention relates to a sequential chromatographic procedure for the purification of a human tumor necrosis factor, produced, by the LuKII cell line, termed TNF(LuKII), the characterization of TNF(LuKII) and hybridoma cell lines producing monoclonal antibodies to TNF(LuKII).
It has been reported that during certain bacterial infections, for example, staphylococcal and streptococcal, there sometimes occurs a concomittant regression of human tumors.
Coley and others treated human malignancies with heat-killed bacterial vaccines and obtained positive results in some patients.
The presence of a tumor inhibitory factor in the sera of mice infected with bacillus Calmette-Guerin (BCG) and subsequently injected with endotoxin was reported by E. A. Carswell et al, Proc. Natl. Acad. Sci. U.S.A., 72, 3666-3670 (1975). This sera has been observed to cause the hemorrhagic necrosis and regression of certain mouse tumors in vivo. This sera was also found to have cytotoxic/cytostatic effects on mouse and human tumor cells in vitro (E. A. Carswell et al, supra; L. Helson, et al, Nature (London), 258, 731-732 (1975); D. N. Mannel et al, Infect. Immun., 28, 204-211 (1980); F. C. Kull and P. Cuatrecasas, J. Immunol., 126, 1279-1283 (1981); K. Haranaka and N. Satomi, Jpn. J. Exp. Med., 51, 191-194 (1981)). A similar factor was found to be induced in rats (Carswell et al, supra) and rabbits (Carswell et al, supra, N. Matthews and J. F Watkins, Br. J. Cancer, 38, 302-309 (1978); J. M. Ostrove and G. E. Gifford, Proc. Soc. Exp. Biol. Med., 160, 354-358 (1979)).
The antitumor factor present in the sera of animals sensitized to BCG or other immunopotentiating agents, such as Corynebacterium parvum, Malaria or Zymosan (yeast cell wall), and then challenged with endotoxin has been termed tumor necrosis factor (TNF).
Biochemical studies have indicated that mouse serum TNF is a glycoprotein and that its activity is associated with both high molecular weight components, e.g., M.sub.r 150,000, (Kull and Cuatrecasas, supra and S. Green et al, Proc. Natl. Acad. Sci U.S.A., 73, 381-385 (1976)) and components in the M.sub.r 40,000-60,000 range, (D. N. Mannel et al, supra; Kull and Cuatrecasas, supra; and Haranaka, supra). The molecule is stable when frozen, preferably below -70.degree. C. Its activity is destroyed at 70.degree. C. for 30 minutes. It is pyrogenic in rabbits in a range from 5-500 microgram/kg and non-pyrogenic at 5 microgram/kg. TNF in rabbit serum has also been reported to have a molecular weight of 39,000, (N. Matthews et al, Br. J. Cancer, 42, 416-422 (1980)), and 67,000, (M. R. Ruff and G. E. Gifford, J. Immunol., 125, 1671-1677 (1980)).
Studies have indicated that both in vivo and in vitro activities of mouse TNF appear to be a property of the same molecule. The cellular source of TNF in the mouse was initially assumed to be the macrophage, because the agents used to prime for TNF production cause massive hyperplasia of macrophages in liver and spleen (Carswell et al, supra). From studies of macrophage-rich cell populations in vitro, (N. Matthews, Br. J. Cancer, 38, 310-315 (1978) and D. N. Mannel et al, Infect. Immun., 30, 523-530 (1980)) a similar conclusion was reached with regard to the source of mouse and rabbit TNF. Direct evidence that macrophages are at least one cell type in the mouse capable of producing TNF comes from studies with cloned lines of mouse histiocytomas (D. N. Mannel et al, supra and unpublished data). These cells constitutively produce low levels of TNF that are greatly increased after exposure to endotoxin.
B. D. Williamson et al, Proc. Natl. Acad. Sci. U.S.A., 80, 5397-5401 (1983), described the capacity of human cell lines of hematopoietic origin, e.g., B-cell lines, to produce a factor with TNF activity. The product of one of the B-cell lines (LuKII) was chosen for detailed studies. Evidence demonstrating that this molecule is a human TNF included the following: (1) the anticellular response of a panel of human cell lines to human TNF, e.g., TNF(LuKII), or mouse TNF are indistinguishable and can be potentiated in a synergistic fashion by interferon, (2) mouse L cells made resistant to mouse TNF are resistant to human TNF, e.g., TNF (LuKII), (3) mouse L cells made resistant to human TNF, e.g., TNF(LuKII) are resistant to mouse TNF , and (4) human TNF, e.g., TNF(LuKII), causes hemorrhagic necrosis of Method A sarcoma in the standard in vivo TNF assay, B. D. Williamson, supra.
Heretofore, there have been no known purification methodologies to obtain pure TNF(LuKII) which are rapid and efficient, for example, which do not require dialysis which may involve serious losses of material. At the present time there is also no hybridoma in existence which produces a monoclonal antibody to the pure TNF(LuKII), which would be extremely useful for purification, diagnostic, and perhaps therapeutic purposes.